The voltage-gated Na channel is an integral membrane protein, part of a macromolecular complex that is central to signaling in the heart and other excitable tissues. Mutations in the channel subunits underlie heritable cardiac arrhythmias, myotonias, epilepsy and autism. Regulation of the essential functions of the channel, namely conduction and gating are complex and differ among the tissue-specific isoforms. A mechanistic understanding of the molecular regulation of the channel is beginning to emerge; however, many of the relevant studies have been limited by the fact that they were performed in expression systems rather than native cells. We and others have demonstrated the importance of the carboxyl terminus (CT) in the regulation of functional expression of the channel. The CT is a hot spot for mutations that produce inherited arrhythmias and possibly cardiomyopathies. Mutations of critical structural motifs in the CT (IQ and EFL) have profound and isoform-specific effects on channel gating, trafficking and drug block. This proposal builds on the work from the prior period of support and will test the following hypotheses: 1. The CT of the Na channel is vitally important in trafficking, gating, Ca2+ sensitivity and functional regulation of the channel. 2. Structural divergence in the CT of different NaV isoforms underlies the functional variance in channel regulation. 3. Altered Na channel trafficking and function with consequent intracellular Na+ overload contributes to the development of dilated cardiomyopathy. The hypotheses will be tested with the following specific aims: 1. Characterize the functional effects, regulation and pharmacology of mutations of the CaM binding IQ motif and Ca2+ binding EFL motif and naturally occurring mutations in the CT of NaV1.5 expressed in neonatal (NRVMs) and adult ventricular myocytes (VM). 2. Characterize the structure function relationships of the proximal CT of NaV1.5 and NaV1.4. 3. Characterization of the contractile and electrophysiological phenotype of a CaM binding deficient knock-in mouse. Given the central role of the Na current in normal physiology and disease this proposal promises to further our understanding of the essential functions of this channel, pathophysiological mechanisms of diseases of excitability and the mechanism of action of clinically important drugs.